1. Field of the Invention
The present invention relates to manually operated presses for use in analyzing characteristics of the liquid content of biological cells.
2. Description of the Prior Art
It has long been desired to obtain the sap from the cells of plant tissues in particular, using various methods. Small hand operated presses have been used. For example, a Micropress Assembly having a base and a fixed barrel with an outlet into which a plunger can be inserted for attempting to squeeze liquid material from tissue is shown in an article entitled Micropress for Obtaining Press Sap from Plant Tissues" by Sreenivasaya et al. in Indian Journal of Experimental Biology, Vol. 5, (April 1967).
Another type of press which is hand operated and moves in a barrel for forcing liquid from plant tissue is shown in a paper entitled A Press for Recovery of Fluids from Plant Tissues by Broyer in Plant Physiology, Vol. 16 pp 419-421 (1941). This device shows a type of a sieve plate at the bottom of the chamber, but the press is quite large in diameter, and has a chamber at the bottom through which sap can be expressed. This device is specifically designed for operation with a screw type press or a hydraulic cylinder press and it is not intended to be hand operated. In other words, the device is of substantial size and requires substantial force to operate.
Another type of press that has a fairly large piston (5 centimeters) is shown in an article entitled Technique D'Obtention Des Sucs Vegetaux by J. Susplugas, et al. in Methodology of Plant Eco-physiology, pages 393-397 (1965).
However rapidly used, reliable presses which are portable and adapted to existing analysis equipment have not been available. The need for analysis of cell sap or other liquids from biological cells continues to exist in order to study cell and plant characteristics, and to obtain favorable characteristics in future generations of plants and animals.
The osmotic potential of plant tissues is an important component of the overall water relations of the plant. As is seen in equation 1 the water potential (.gamma.w) of a cell is equal to the osmotic potential (.gamma.s) plus the turgor potential (.gamma.p). EQU .gamma.w=.gamma.s+.gamma.p (1)
Equation 1 can be rearranged to show that the turgor potential of the tissue is proportional to the osmotic potential at a constant water potential (eq. 2). EQU .gamma.p.alpha..gamma.s*(constant .gamma.w) (2)
The osmotic potential therefore is extremely important in determining the turgor of the leaf. The lower the osmotic potential, the lower the water potential the plant can reach and still have positive turgor. The point of zero turgor has been associated with wilting, decreased photosynthesis, and reduced growth in many plant species. Recently it has been observed that many plant species show changes in osmotic potential when exposed to periods of water stress. These observations suggest that the maintenance of a positive turgor in plants is an important adaptation to growth under drought conditions.
Although there are excellent instruments available for determining the osmotic potential of solutions, there remain considerable problems in accurately measuring the osmotic potential of bulk tissue. The limitations are due primarily to inadequate methods of extraction of sap from tissue cells and subsequent transfer of the sap to the measuring instrument without changes in concentration.
The osmotic potential of the sap is presently carried out with very reliable instruments that can measure the osmotic potential of the plant cell with small amounts of cell sap, sufficient only to wet a small filter paper. In order to obtain cell sap, the cell membranes (the lining just inside the cell wall) have to be ruptured. One method that is commonly used to rupture the cell membranes is to freeze the tissue and then thaw it again. The expanding ice crystals upon freezing pierce the cell membrane and the cell walls, eliminating any cell tugor, which is the rigidity or turgidity of the cell. The thawed piece of leaf tissue is then placed in a thermocouple psychrometer (a conventional instrument) and the water potential of the dead tissue is measured.
A second method for obtaining cell sap is to place the tissue in a cylinder with a mesh covered hole at one end, much like the pressures shown in the previously described prior art, and then compressing the tissue by a plunger moving down the cylinder barrel. This generally requires a large press with existing equipment, and the resulting leaf sap is forced out a small hole in the end of the barrel and collected for future analysis. Large pieces of cell material that pass through the screen are removed by either centrifugation or filtration, and then the remaining solution is tested in an osmometer for water potential.
In the first method, after the sample has been put into a thermocouple instrument, an equilibrium time of at least two hours is needed before an accurate measurement can be obtained. This requires that a large number of thermocouple points be used. Twelve to Twenty Four such points are required for adequate replication. Each point requires its own calibration procedure which requires five solutions of known osmotic potential.
During the length of time needed for insuring that equilibrium conditions have been established, there is ample opportunity for enzyme action to degrade the tissue components causing a change in the osmotic potential of the sap being tested. Recent studies have shown that such degradation does occur.
The second method described, which uses a vapor pressure osmometer for measuring the osmotic potential of the extracted solution, has the advantage of easy calibration and a very small sample size. Equilibrium time is extremely rapid, and the measurement can be completed in only ninety seconds. The limitations of using this method, however, lie in the sap extraction procedure. One disadvantage of a conventional cylinder (press) extraction is that changes can occur in the osmotic potential of the expressed cell sap between extraction and measurement. Any exposure of the sap to the atmosphere allows evaporation of water and a change of the osmotic potential of the sap. Centrifuging and filtering both require time and can result in changes in the osmotic potential of the sap. The procedure is time consuming and thus tedious.